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Solution A

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Internal Solution-ID: S4684

Equipment needed: Anaerobic workbench Filter Autoclave
1 First dissolve iron(III) citrate in 800 ml distilled water and adjust pH to 7.0 with 10 N NaOH. Then, add the remaining minerals, bring volume to 970 ml and autoclave under a N2 atmosphere. Add aseptically and anaerobically the following filter-sterilized solutions:
Compound Amount Unit Conc. [g/L] Conc. [mM]
Iron(III) citrate 12.00 g 333.333 1360.88
NaCl 1.00 g 27.778 475.321
KCl 0.50 g 13.889 186.303
MgCl2 x 6 H2O 0.40 g 11.111 54.653
NH4Cl 0.25 g 6.944 129.827
KH2PO4 0.20 g 5.556 40.824
CaCl2 x 2 H2O 0.10 g 2.778 18.895
Na2SO4 1.42 mg 0.039 0.278
Sodium acetate x 3 H2O 1.36 g 37.778 -
NaHCO3
(8.5%) 		30.00 ml - -
FeCl2 solution 1.00 ml - -
Trace element solution (see Medium No. 187 1.00 ml - -
Vitamin solution 1.00 ml - -
Thiamine solution 1.00 ml - -
Vitamin B12 solution 0.05 g/L 1.00 ml - -
Selenite-tungstate solution 1.00 ml - -
2 Distribute the medium into sterile culture vessels (e.g., 50 ml per 120 ml bottle) under a N2-CO2 (4:1, v/v) gas mixture, and seal with sterile butyl rubber stoppers. Then add per 50 ml the following solutions from anaerobic stocks:
FeCl2
(100 mM) 		0.50 ml - -
Toluene 5.00 �l - -
3 Check the final pH of the medium to be around 7.0.
J747 FRESHWATER MEDIUM WITH TOLUENE